HDL-bound Sphingosine 1-phosphate Research Articles

Designer high-density lipoprotein particles enhance endothelial barrier function and suppress inflammation.

High-density lipoprotein (HDL) nanoparticles promote endothelial cell (EC) function and suppress inflammation, but their utility in treating EC dysfunction has not been fully explored. Here, we describe a fusion protein named ApoA1-ApoM (A1M) consisting of apolipoprotein A1 (ApoA1), the principal structural protein of HDL that forms lipid nanoparticles, and ApoM, a chaperone for the bioactive lipid sphingosine 1-phosphate (S1P). A1M forms HDL-like particles, binds to S1P, and is signaling competent. Molecular dynamics simulations showed that the S1P-bound ApoM moiety in A1M efficiently activated EC surface receptors. Treatment of human umbilical vein ECs with A1M-S1P stimulated barrier function either alone or cooperatively with other barrier-enhancing molecules, including the stable prostacyclin analog iloprost, and suppressed cytokine-induced inflammation. A1M-S1P injection into mice during sterile inflammation suppressed neutrophil influx and inflammatory mediator secretion. Moreover, systemic A1M administration led to a sustained increase in circulating HDL-bound S1P and suppressed inflammation in a murine model of LPS-induced endotoxemia. We propose that A1M administration may enhance vascular endothelial barrier function, suppress cytokine storm, and promote resilience of the vascular endothelium.

The effect of normobaric hypoxia on acute exercise-induced changes in blood sphingoid base-1-phosphates metabolism in cyclists.

Extracellular sphingosine-1-phosphate (S1P) emerged as an important regulator of muscle function. We previously found that plasma S1P concentration is elevated in response to acute exercise and training. Interestingly, hypoxia, which is commonly utilized in training programs, induces a similar effect. Therefore, the aim of the current study was to determine the effect of normobaric hypoxia on exercise-induced changes in blood sphingolipid metabolism. Fifteen male competitive cyclists performed a graded cycling exercise until exhaustion (GE) and a simulated 30 km individual time trial (TT) in either normoxic or hypoxic (FiO2 = 16.5%) conditions. Blood samples were taken before the exercise, following its cessation, and after 30 min of recovery. We found that TT increased dihydrosphingosine-1-phosphate (dhS1P) concentration in plasma (both HDL- and albumin-bound) and blood cells, as well as the rate of dhS1P release from erythrocytes, regardless of oxygen availability. Plasma concentration of S1P was, however, reduced during the recovery phase, and this trend was augmented by hypoxia. On the other hand, GE in normoxia induced a selective increase in HDL-bound S1P. This effect disappeared when the exercise was performed in hypoxia, and it was associated with reduced S1P level in platelets and erythrocytes. We conclude that submaximal exercise elevates total plasma dhS1P concentration via increased availability of dihydrosphingosine resulting in enhanced dhS1P synthesis and release by blood cells. Maximal exercise, on the other hand, induces a selective increase in HDL-bound S1P, which is a consequence of mechanisms not related to blood cells. We also conclude that hypoxia reduces post-exercise plasma S1P concentration.

Therapeutic activation of endothelial sphingosine-1-phosphate receptor 1 by chaperone-bound S1P suppresses proliferative retinal neovascularization.

Sphingosine-1-phosphate (S1P), the circulating HDL-bound lipid mediator that acts via S1P receptors (S1PR), is required for normal vascular development. The role of this signaling axis in vascular retinopathies is unclear. Here, we show in a mouse model of oxygen-induced retinopathy (OIR) that endothelial overexpression of S1pr1 suppresses while endothelial knockout of S1pr1 worsens neovascular tuft formation. Furthermore, neovascular tufts are increased in Apom-/- mice which lack HDL-bound S1P while they are suppressed in ApomTG mice which have more circulating HDL-S1P. These results suggest that circulating HDL-S1P activation of endothelial S1PR1 suppresses neovascular pathology in OIR. Additionally, systemic administration of ApoM-Fc-bound S1P or a small-molecule Gi-biased S1PR1 agonist suppressed neovascular tuft formation. Circulating HDL-S1P activation of endothelial S1PR1 may be a key protective mechanism to guard against neovascular retinopathies that occur not only in premature infants but also in diabetic patients and aging people.

Interplay between S1P receptors and SR-BI in atherosclerosis relevant cells: New insight from transgenic animals

Background and Aims : Sphingosine 1-phosphate (S1P), traveling in plasma mainly bound to high-density lipoproteins (HDL), fulfills several tasks in immune and cardiovascular systems by binding to its G protein-coupled receptors (S1P1-5). SR-BI, an HDL receptor, is widely expressed in different cell types, including endothelial cells and macrophages, and plays key roles in cholesterol homeostasis and lipoprotein metabolism. Recent evidence showed that HDL-bound S1P stimulates the transient interaction between SR-BI and S1PRs, activating S1PRs.

Sphingosine-1-phosphate: metabolism, transport, atheroprotection and effect of statin treatment.

To better define the metabolism of sphingosine-1-phosphate (S1P), its transport in plasma and its interactions with S1P receptors on vascular cells, and to evaluate the effect of statin treatment on the subnormal plasma levels of high-density lipoprotein (HDL)-bound S1P characteristic of the atherogenic dyslipidemia of metabolic syndrome (MetS). Neither clinical intervention trials targeted to raising high-density lipoprotein-cholesterol (HDL-C) levels nor human genome-wide association studies (GWAS) studies have provided evidence to support an atheroprotective role of HDL. Recently however a large monogenic univariable Mendelian randomization on the N396S mutation in the gene encoding endothelial lipase revealed a causal protective effect of elevated HDL-C on coronary artery disease conferred by reduced enzyme activity. Given the complexity of the HDL lipidome and proteome, components of HDL other than cholesterol may in all likelihood contribute to such a protective effect. Among HDL lipids, S1P is a bioactive sphingolipid present in a small proportion of HDL particles (about 5%); indeed, S1P is preferentially enriched in small dense HDL3. As S1P is bound to apolipoprotein (apo) M in HDL, such enrichment is consistent with the elevated apoM concentration in HDL3. When HDL/apoM-bound S1P acts on S1P1 or S1P3 receptors in endothelial cells, potent antiatherogenic and vasculoprotective effects are exerted; those exerted by albumin-bound S1P at these receptors are typically weaker. When HDL/apoM-bound S1P binds to S1P2 receptors, proatherogenic effects may potentially be induced. Subnormal plasma levels of HDL-associated S1P are typical of dyslipidemic individuals at high cardiovascular risk and in patients with coronary heart disease. International Guidelines recommend statin treatment as first-line lipid lowering therapy in these groups. The cardiovascular benefits of statin therapy are derived primarily from reduction in low-density lipoprotein (LDL)-cholesterol, although minor contributions from pleiotropic actions cannot be excluded. Might statin treatment therefore normalize, directly or indirectly, the subnormal levels of S1P in dyslipidemic subjects at high cardiovascular risk? Our unpublished findings in the CAPITAIN study (ClinicalTrials.gov: NCT01595828), involving a cohort of obese, hypertriglyceridemic subjects (n = 12) exhibiting the MetS, showed that pitavastatin calcium (4 mg/day) treatment for 180days was without effect on either total plasma or HDL-associated S1P levels, suggesting that statin-mediated improvement of endothelial function is not due to normalization of HDL-bound S1P. Statins may however induce the expression of S1P1 receptors in endothelial cells, thereby potentiating increase in endothelial nitric oxide synthase response to HDL-bound S1P, with beneficial downstream vasculoprotective effects. Current evidence indicates that S1P in small dense HDL3 containing apoM exerts antiatherogenic effects and that statins exert vasculoprotective effects through activation of endothelial cell S1P1 receptors in response to HDL/apoM-bound S1P.

Endothelial Transporter Spinster 2 (SPNS2) and Apolipoprotein M (ApoM) Regulation of Vascular Tone and Hypertension via Sphingosine‐1‐phosphate (S1P)

Most of the circulating sphingosine-1-phosphate (S1P) is bound to ApoM of HDL and mediates many beneficial effects of HDL on the vasculature via G protein coupled S1P receptors. Decreased plasma levels of HDL-bound S1P are associated with atherosclerosis, myocardial infarction and diabetes. In addition to be the target, the endothelium is a source of S1P, which is transported outside of the cells by Spinster-2 (SPNS2), contributing to the circulating S1P pool as well as to local signaling. Mice lacking endothelial S1P receptor 1 are hypertensive, suggesting a vasculo-protective role of S1P signaling. Whether locally produced S1P differentially regulate vascular tone and blood pressure (BP) vs. circulating S1P is not known and has been investigated here. ApoM knockout mice (ApoM KO) and mice lacking endothelial Spns-2 (ECKO-Spns2) were infused with angiotensin II (AngII) for 28 days. BP, measured by telemetry and tail-cuff, was significantly increased in both ECKO-Spns2 and ApoM KO vs. control mice, at baseline and following AngII. Notably, ECKO-Spns2 presented an impaired BP dipping, which is clinically associated to increased risk for cardiovascular events. In hypertension both groups presented reduced flow-mediated vasodilation and some degree of impairment in endothelial nitric oxide (NO) production, which was more evident in ECKO-Spns2. Increased hypertension in ECKO-Spns2 and ApoM KO mice correlated with worsen cardiac hypertrophy vs. controls. Our study identifies an important role for SPNS2 and ApoM-HDL in BP homeostasis via S1P-NO signaling and dissects the pathophysiological impact of locally produced versus ApoM of HDL-bound S1P in hypertension and cardiac hypertrophy.

Novel Insights into the Role of HDL-Associated Sphingosine-1-Phosphate in Cardiometabolic Diseases.

Sphingolipids are key signaling molecules involved in the regulation of cell physiology. These species are found in tissues and in circulation. Although they only constitute a small fraction in lipid composition of circulating lipoproteins, their concentration in plasma and distribution among plasma lipoproteins appears distorted under adverse cardiometabolic conditions such as diabetes mellitus. Sphingosine-1-phosphate (S1P), one of their main representatives, is involved in regulating cardiomyocyte homeostasis in different models of experimental cardiomyopathy. Cardiomyopathy is a common complication of diabetes mellitus and represents a main risk factor for heart failure. Notably, plasma concentration of S1P, particularly high-density lipoprotein (HDL)-bound S1P, may be decreased in patients with diabetes mellitus, and hence, inversely related to cardiac alterations. Despite this, little attention has been given to the circulating levels of either total S1P or HDL-bound S1P as potential biomarkers of diabetic cardiomyopathy. Thus, this review will focus on the potential role of HDL-bound S1P as a circulating biomarker in the diagnosis of main cardiometabolic complications frequently associated with systemic metabolic syndromes with impaired insulin signaling. Given the bioactive nature of these molecules, we also evaluated its potential of HDL-bound S1P-raising strategies for the treatment of cardiometabolic disease.

Abstract 069: Local and Circulating Sphingosine-1-Phosphate in Blood Pressure Regulation

Sphingosine-1-phosphate (S1P), a bioactive lipid, regulates different biological processes in health and disease. Once synthetized, endothelial S1P is transported outside the cells via Spinster-2 (Spns-2), and can signal mainly through the sphingosine-1-phosphate receptor-1 (S1P1) in autocrine/paracrine manner. Alternatively, S1P can bind circulating carriers, including ApoM of HDL or albumin. Recently, we reported a key role of endothelial S1P-S1P1 autocrine signaling in vascular function and blood pressure (BP) homeostasis. To dissect the contribution of endothelium-derived S1P versus circulating HDL-bound S1P on BP regulation, we used ApoM knockout mice (ApoM-/-) and developed mice lacking the endothelial Spinster-2 (ECKO-Spns2). At baseline, systolic BP (SBP) was markedly increased in both ECKO-Spns2 (120.8± 2.1 vs. 106.6±1.3 mmHg, n=8) and ApoM-/- (122.6±1.0 vs. 109.0±1.4 mmHg, n=8) vs control mice. Vasodilation of mesenteric arteries (MA) to acetylcholine was preserved in both mouse models, whereas flow-induced vasodilation was significantly reduced in ECKO-Spns2 (Emax 36.0±1.5 vs. 53.6±3.6 % vasodilation, n=5) but not in ApoM-/- MA, suggesting that autocrine S1P signaling requires the S1P transporter Spns-2. However, both ECKO-Spns2 and ApoM-/- showed a reduced NO production compared to control MA. Moreover, chronic infusion of AngII resulted in higher BP in ECKO-Spns2 (156.0± 1.2 vs. 142.0±1.6 mmHg, n=8) and ApoM-/- (158.5±1.1 vs. 144.1±1.3 mmHg, n=8) vs control mice. Interestingly, chronic AngII strongly reduced vasodilation in response to flow in both ECKO-Spns2 (Emax 19.3±2.5 vs. 35.4±7.1 % vasodilation, n=5) and ApoM-/- (Emax 22.9±1.1 vs. 34.8±6.8 % vasodilation, n=5) compared to control MA, suggesting a protective role of local and circulating S1P. Additionally, basal NO production was further impaired by the AngII infusion (ECKO-Spns2 -32.7% and ApoM-/- -42.8%, vs control MA), confirming the pivotal role of S1P-S1P1-NO pathway in vascular and BP homeostasis.

Arteriovenous Sphingosine-1-Phosphate Differences Across Selected Organs of the Rat

Background/Aims: Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid that is found in high concentration in plasma. The majority of plasma S1P is transported bound to HDL and albumin. Although the major sources of circulating S1P have been identified, it remains obscure what is the contribution of different organs/tissues to S1P homeostasis in plasma. Answering this question was the major aim of the present study. Methods: The experiment was performed on male Wistar rats from whom blood samples were taken from either: 1) femoral vein, right ventricle of the heart, and abdominal aorta (n=15) or 2) hepatic vein, portal vein, and abdominal aorta (n=11). Plasma was fractionated by sequential flotation ultracentrifugation and sphingolipids were quantified by a HPLC method. Results: Compared to the mixed venous blood sampled from the right ventricle, total plasma and lipoprotein-depleted plasma (LPDP) concentration of S1P in the arterial blood was lower. On the other hand, the level of S1P increased across the leg both in plasma and LPDP. The concentration of S1P, sphingosine, and sphinganine in the plasma, HDL, and LPDP isolated from the blood taken from the hepatic vein was markedly higher compared to both arterial and portal blood. Conclusions: We conclude that, in contrast to HDL-bound S1P, albumin-associated S1P is very labile in the circulation. It is degraded in the pulmonary, and to a lesser extent, gastrointestinal circulation, and released across the liver and skeletal muscle. We also conclude that liver is an important source of HDL-bound S1P and circulating free sphingoid bases.

Endurance training selectively increases high-density lipoprotein-bound sphingosine-1-phosphate in the plasma.

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid that is found in relatively high concentration in human plasma. Erythrocytes, endothelial cells, and activated platelets are the main sources of circulating S1P. The majority of plasma S1P is transported bound to high-density lipoprotein (HDL) and albumin. In recent years, HDL-bound S1P attracted much attention due to its cardioprotective and anti-atherogenic properties. We have previously found that endurance-trained athletes are characterized by higher plasma S1P concentration compared to untrained individuals. This finding prompted us to examine the effect of endurance training on S1P metabolism in blood. Thirteen healthy, untrained, male subjects completed an 8-week training program on a rowing ergometer. Three days before the first, and 3days after the last training session, blood samples were drawn from an antecubitalvein. We found that total plasma S1P concentration was increased after the training. Further analysis of different plasma fractions showed that the training selectively elevated HDL-bound S1P. This effect was associated with activation of sphingosine kinase in erythrocytes and platelets and enhanced S1P release from red blood cells. We postulate that increase in HDL-bound S1P level is one of the mechanisms underlying beneficial effects of regular physical activity on cardiovascular diseases.

Erratum for the Research Article: “HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P 1 to limit vascular inflammation” by S. Galvani, M. Sanson, V. A. Blaho, S. L. Swendeman, H. Conger, B. Dahlbäck, M. Kono, R. L. Proia, J. D. Smith, T. Hla

An author is added to a Research Article.

HDL-bound sphingosine 1-phosphate acts as a biased agonist for the endothelial cell receptor S1P1 to limit vascular inflammation.

The sphingosine 1-phosphate receptor 1 (S1P1) is abundant in endothelial cells, where it regulates vascular development and microvascular barrier function. In investigating the role of endothelial cell S1P1 in adult mice, we found that the endothelial S1P1 signal was enhanced in regions of the arterial vasculature experiencing inflammation. The abundance of proinflammatory adhesion proteins, such as ICAM-1, was enhanced in mice with endothelial cell-specific deletion of S1pr1 and suppressed in mice with endothelial cell-specific overexpression of S1pr1, suggesting a protective function of S1P1 in vascular disease. The chaperones ApoM(+)HDL (HDL) or albumin bind to sphingosine 1-phosphate (S1P) in the circulation; therefore, we tested the effects of S1P bound to each chaperone on S1P1 signaling in cultured human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to ApoM(+)HDL-S1P, but not to albumin-S1P, promoted the formation of a cell surface S1P1-β-arrestin 2 complex and attenuated the ability of the proinflammatory cytokine TNFα to activate NF-κB and increase ICAM-1 abundance. Although S1P bound to either chaperone induced MAPK activation, albumin-S1P triggered greater Gi activation and receptor endocytosis. Endothelial cell-specific deletion of S1pr1 in the hypercholesterolemic Apoe(-/-) mouse model of atherosclerosis enhanced atherosclerotic lesion formation in the descending aorta. We propose that the ability of ApoM(+)HDL to act as a biased agonist on S1P1 inhibits vascular inflammation, which may partially explain the cardiovascular protective functions of HDL.

HDL-Bound Sphingosine 1-Phosphate (S1P) Predicts the Severity of Coronary Artery Atherosclerosis

Background: We have recently demonstrated a reduction in HDL-bound sphingosine 1-phosphate (S1P) in patients with stable coronary artery disease (CAD). In the current study, we tested whether HDL-associated S1P is predictive for the degree of coronary stenosis, restenosis and overall CAD severity on follow up in patients undergoing elective percutaneous coronary intervention (PCI). Methods: Coronary angiography of patients with CAD (n=59) undergoing elective PCI and presenting for a follow up after 6 months (n=48) was graded for disease severity defined clinically as 1- or multi-vessel disease. Target lesion stenosis was quantified by quantitative coronary angiography (QCA). S1P in plasma and isolated HDL were measured by mass spectrometry in the initial samples and in 32 available follow up samples. Results: HDL-bound S1P levels remained stable over time and correlated closely at first visit and follow up. While not associated with the extent of target lesion stenosis or restenosis, HDL-bound S1P correlated negatively with the overall severity of CAD and discriminated 1-vessel-disease from multi-vessel disease. Furthermore, low HDL-bound S1P was predictive for CAD extent. Conclusion: In stable CAD, HDL-bound S1P does not predict the degree of stenosis or restenosis of the target lesion but constitutes a marker of clinically defined disease burden.

S1P control of endothelial integrity.

Sphingosine 1-phosphate (S1P), a lipid mediator produced by sphingolipid metabolism, promotes endothelial cell spreading, vascular maturation/stabilization, and barrier function. S1P is present at high concentrations in the circulatory system, whereas in tissues its levels are low. This so-called vascular S1P gradient is essential for S1P to regulate much physiological and pathophysiological progress such as the modulation of vascular permeability. Cellular sources of S1P in blood has only recently begun to be identified. In this review, we summarize the current understanding of S1P in regulating vascular integrity. In particular, we discuss the recent discovery of the endothelium-protective functions of HDL-bound S1P which is chaperoned by apolipoprotein M.

Sphingosine-1-Phosphate-Specific G Protein-Coupled Receptors as Novel Therapeutic Targets for Atherosclerosis

Atherosclerosis is a chronic inflammatory process involving complex interactions of modified lipoproteins, monocyte-derived macrophages or foam cells, lymphocytes, endothelial cells (ECs), and vascular smooth muscle cells. Sphingosine-1-phosphate (S1P), a biologically active blood-borne lipid mediator, exerts pleiotropic effects such as cell proliferation, migration and cell-cell adhesion in a variety of cell types via five members of S1P-specific high-affinity G protein-coupled receptors (S1P1-S1P5). Among them, S1P1, S1P2 and S1P3 are major receptor subtypes which are widely expressed in various tissues. Available evidence suggest that S1P and HDL-bound S1P exert atheroprotective effects including inhibition of leukocyte adhesion and stimulation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) through the activation of Gi signaling pathway via S1P3 and probably S1P1, although there is still controversy. FTY720, the phosphorylation product of which is a high-affinity agonist for all S1P receptors except S1P2 and act as an immunosuppressant by downregulating S1P1 on lymphocytes, inhibits atherosclerosis in LDL receptor-null mice and apoE-null mice through the inhibition of lymphocyte and macrophage functions and probably stimulation of EC functions, without influencing plasma lipid concentrations. In contrast to S1P1 and S1P3, S1P2 facilitates atherosclerosis by activating G12/13-Rho-Rho kinase (ROCK) in apoE-null mice. S1P2 mediates transmigration of monocytes into the arterial intima, oxidized LDL accumulation and cytokine secretion in monocyte-derived macrophages, and eNOS inhibition and cytokine secretion in ECs through Rac inhibition, NF-κB activation and 3′-specific phosphoinositide phosphatase (PTEN) stimulation downstream of G12/13-Rho-ROCK. Systemic long-term administration of a selective S1P2-blocker remarkably inhibits atherosclerosis without overt toxicity. Thus, multiple S1P receptors positively and negatively regulate atherosclerosis through multitudes of mechanisms. Considering the essential and multi-faceted role of S1P2 in atherogenesis and the impact of S1P2 inactivation on atherosclerosis, S1P2 is a particularly promising therapeutic target for atherosclerosis.

Sphingosine 1-phosphate levels in plasma and HDL are altered in coronary artery disease

High-density lipoproteins (HDL) are the major plasma carriers for sphingosine 1-phosphate (S1P) in healthy individuals, but their S1P content is unknown for patients with coronary artery disease (CAD). The aim of the study was to determine whether the S1P levels in plasma and HDL are altered in coronary artery disease. S1P was determined in plasma and HDL isolated by ultracentrifugation from patients with myocardial infarction (MI, n = 83), stable CAD (sCAD, n = 95), and controls (n = 85). In our study, total plasma S1P levels were lower in sCAD than in controls (305 vs. 350 pmol/mL). However, normalization to HDL-cholesterol (a known determinant of plasma S1P) revealed higher normalized plasma S1P levels in sCAD than in controls (725 vs. 542 pmol/mg) and even higher ones in MI (902 pmol/mg). The S1P amount contained in isolated HDL from these individuals was lower in sCAD than in controls (S1P per protein in HDL: 132 vs. 153 pmol/mg). The amount of total plasma S1P bound to HDL was lower in sCAD and MI than in controls (sCAD: 204, MI: 222, controls: 335 pmol/mL), while the non-HDL-bound S1P was, accordingly, higher (sCAD: 84, MI: 81, controls: 10 pmol/mL). HDL-bound plasma S1P was dependent on the plasma HDL-C in all groups, but normalization to HDL-C still yielded lower HDL-bound plasma S1P in patients with sCAD than in controls (465 vs. 523 pmol/mg). The ratio of non-HDL-bound plasma S1P to HDL-C-normalized HDL-bound S1P was also higher in both sCAD (0.18 mg/mL) and MI (0.15 mg/mL) than in controls (0.02 mg/mL). Remarkably, levels of non-HDL-bound plasma S1P correlated with the severity of CAD symptoms as graded by Canadian Cardiovascular Score, and discriminated patients with MI and sCAD from controls. Furthermore, a negative association was present between non-HDL-bound plasma S1P and the S1P content of isolated HDL in controls, but was absent in sCAD and MI. Finally, MI patients with symptom duration of less than 12 h had the highest levels of total and normalized plasma S1P, as well as the highest levels of S1P in isolated HDL. The HDL-C-normalized plasma level of S1P is increased in sCAD and even further in MI. This may be caused by an uptake defect of HDL for plasma S1P in CAD, and may represent a novel marker of HDL dysfunction.